a) Field of the Invention
The present invention is concerned with a human protein called “HIF-3α” that is a Hypoxia Inducible Factor-3α and more particularly to the use of HIF-3α nucleic acids, proteins, fragments, antibodies, probes, and cells, to characterize HIF-3α, and modulate its cellular levels.
The present invention is also concerned with the use of nucleotide sequences encoding for proteins from the hypoxia inducible factors family for inducing VEGF expression, for inducing angiogenesis and for improving muscular functions, and more particularly, for treating coronary and cardiac diseases in mammals.
b) Brief Description of the Prior Art
Chronic ischemic heart disease is a worldwide health problem of major proportions. According to the American Heart Association, 61 800 000 Americans have at least one type of cardiovascular disease. In particular, coronary heart disease (CHD) cause myocardial infarction (MI) for 7 500 000 American patients and congestive heart failure (CHF) for 4 800 000 American patients. Almost 450 000 deaths in the United States alone were deemed to derive from CHD.
Current CHD treatments include medication, percutaneous transluminal coronary angioplasty and coronary artery bypass surgery. These procedures are quite successful to increase blood flow in the myocardium thus reducing ischemia and ameliorating the condition of the patient. However, due to the progressive nature of CHD, the beneficial effects of these procedures are not permanent and new obstructions can occur. Patients that live longer through effective cardiovascular interventions eventually run out of treatment options. Also an important patient population is still refractory to these treatments due to diffuse athereosclerotic diseases and/or small caliber arteries.
Severe and chronic ischemia can cause MI which is an irreversible scarring of the myocardium. This scarring reduces heart contractility and elasticity and consequently the pumping function, which can then lead to CHF. Treatments available to CHF patients target kidney function and peripheral vasculature to reduce the symptoms but none are treating the scar or increasing pump function of the heart. A very promising approach for reducing the scar and improving heart function is named cellular cardiomyoplasty (CCM). It consists in the injection of cells in the scar, replacing the fibrotic scar by healthy tissue and increasing elasticity (see U.S. Pat. No. 5,130,141; No. 5,602,301, No. 6,099,832 and No. 6,110,459).
Another emerging treatment for CHF patients is therapeutic angiogenesis. Angiogenesis is defined as blood vessel sprouting and proliferation from pre-existing vasculature. The net result is a higher capillary density and better blood perfusion. For instance, U.S. Pat. No. 5,792,453 disclose a method for promoting coronary collateral vessel development by delivering an adenovirus vector with a transgene encoding for an angiogenic protein. Although stimulation of angiogenesis can improve function of ischemic myocardium, it will have no effect on scar tissue because no viable cells will benefit from the improved perfusion.
Many growth factors are currently used to induce angiogenesis, including Vascular Endothelial Growth Factor (VEGF) and Fibroblast Growth Factor (FGF), but none of these factors has the property to stimulate every step of angiogenesis (basal membrane disruption, endothelial cell proliferation, migration and differentiation followed by periendothelial cells recruitment). Since it is known that cell hypoxia can naturally induce a strong angiogenesis, the use of regulators of hypoxia could stimulate the synthesis of one or many angiogenic factors at once, thereby resulting in a more structured and stronger angiogenesis than with individual factors.
Hypoxia Inducible Factors (HIFs) are heterodimeric transcription factors that regulate a number of adaptive responses to low oxygen tension. They are composed of alpha- and beta-subunits that belong to the basic helix-loop-helix-PAS (bHLH-PAS) superfamily. Members of this family include HIF-1α (also known as MOP1; see Wang et al., Proc. Natl. Aca. Sci. USA (1995) 92:5510-5514; and U.S. Pat. Nos. 5,882,314; 6,020,462 and 6,124,131), HIF-2α (also known as Endothelial PAS 1 (EPAS1), MOP2, HIF-related factor (HRF) and HLF (HIF-like factor), see Tian et al., Genes & Dev. (1996) 11:72-82; and U.S. Pat. No. 5,695,963).
Another member of the HIF family has been discovered recently, namely HIF-3α. The cloning of HIF-3α has been described in mice (Gu et al., Gene Expression (1998) 7:205-213; and in International PCT application WO 99/28264) and in rat (Kietzmann et al., Biochem J. (2001), 354 (Pt3):531-537). A partial cDNA sequence of human HIF-3α has been published in 1999 (GenBank™ accession No. AF079154), and a full length sequence of a human HIF-3α isoform, different from the one of the present invention, was published in October 2001 by Hara et al. (Biochem. Biophys. Res. Comm. (2001), October 5; 287:808-813).
HIFs are highly labile in normal conditions, but are stabilized in response to low oxygen tension. This stabilization allows them to bind to cis DNA elements of target genes, and stimulate transcription of hypoxia induced genes that help cell survival in low oxygen conditions. These target genes are implicated in processes such as anaerobic metabolism (glucose transporters and glycolytic enzymes), vasodilatation (inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1)), increased breathing (tyrosine hydroxylase), erythropoiesis (erythropoietin) and angiogenesis (VEGF). Gene activation by HIF-1α or HIF-2α was demonstrated by co-transfection assays, in which a reporter gene is activated by the co-transfected HIF factor (Tian et al., Genes & Dev. (1996) 11: 72-82; Jiang et al., J. Biol. Chem. (1995) 272: 19253-19260). The role of HIF-2α in VEGF activation was also demonstrated in renal cell carcinoma (Xia et al., Cancer (2001), 91:1429-1436). In animal models, strong angiogenesis was reported following gene transfer of a hybrid HIF-1α/VP16 DNA construct (Vincent et al., Circulation (2000) 102: 2255-2261). However, prior to the present invention, it has never been demonstrated or suggested that HIF-2α or HIF-3α could induce the expression of angiogenesis-related gene(s) in mammalian muscular cells, nor that they could induce angiogenesis in these cells. It was also unknown that expression of HIF-1α, HIF-2α or HIF-3α in ischemic muscular tissue resulted in an increased metabolic activity of this tissue, indicating improved function.
Given that HIFs seem to represent ideal factors for VEGF activation and/or for induce angiogenesis, there is thus a need to identify a novel member of the HIF family. There is more particularly a need for a human HIF-3α protein and a nucleic acid encoding the same.
Also, it would be highly desirable to be provided with methods, compositions and cells for inducing angiogenesis and for improving muscular functions.
The present invention fulfils these needs and also other needs as it will be apparent to those skilled in the art upon reading the following specification.